Solventless extraction process

ABSTRACT

The present invention provides a method for extracting lipids from microorganisms without using organic solvent as an extraction solvent. In particular, the present invention provides a method for extracting lipids from microorganisms by lysing cells and removing water soluble compound and/or materials by washing the lysed cell mixtures with aqueous washing solutions until a substantially non-emulsified lipid is obtained.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior pending U.S. applicationSer. No. 10/784,148, filed Feb. 20, 2004, and which issued as U.S. Pat.No. 7,351,558 on Apr. 1, 2008; which is a continuation of U.S.application Ser. No. 09/766,500, filed Jan. 19, 2001, and which issuedas U.S. Pat. No. 6,750,048 on Jun. 15, 2004; which claims the benefit ofpriority under 35 U.S.C. §119(e) from Provisional Patent ApplicationSer. No. 60/177,125, filed on Jan. 19, 2000. Each of the foregoingapplication is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to a process for extracting lipidsfrom microorganisms without the use of any significant amount of anorganic solvent.

BACKGROUND OF THE INVENTION

A typical microorganism lipid manufacturing process, such as productionof omega-3 highly unsaturated fatty acid, in particular docosahexaenoicacid (DHA), involves growing microorganisms which are capable ofproducing the desired lipid in a fermentor, pond or bioreactor,isolating the microbial biomass, drying it, and extracting intracellularlipids with an organic solvent, e.g., hexane. Generally, intracellularlipids of microorganisms are extracted after rupturing (i.e., lysing)the cells of the microorganisms. The extracted lipids are can be furtherrefined to produce a high purity and/or quality lipids. Themicroorganisms are generally isolated by first diluting the fermentationbroth with water, and centrifuging the mixture to isolatemicroorganisms. When lipids are not extracted immediately or soon afterisolating the microorganisms, the isolated microorganisms are typicallydried, for example, on a drum dryer, and packaged, for example, invacuum-sealed bags, to prevent degradation of lipids.

Unfortunately, the drying process exposes the microorganisms to heat,which can damage, i.e., degrade the quality of, lipids if doneincorrectly. The vacuum-sealed bags may develop leaks, which can furtherdegrade the quality of the lipids due to exposure of the microorganismsto air. In addition, if the dried microorganisms are not treated with anantioxidant, lipids can be further degraded due to exposure to air, forexample, DHA may degrade due to oxidation by air. Furthermore, in somecases operators who are exposed to the dried microorganisms can developan allergic reaction creating a safety and/or health hazard tooperators.

Moreover, in an industrial scale production, the amount of organicsolvent used in lipid extraction typically requires a large amount ofvolatile and flammable organic solvent, thereby creating hazardousoperating conditions. The use of organic solvent in the extractionprocess may necessitate using an explosion-proof oil recovery system,thereby adding to the cost of lipid recovery. Moreover, use of anorganic solvent in extracting lipids from microorganisms generate anorganic solvent waste stream requiring a proper method, which furtherincreases the overall production cost of lipid extraction.

Therefore, there is a need for a process for extracting lipids frommicroorganisms which does not require the use of an organic solvent.There is also a need for a lipid extraction process from microorganismswhich does not require the expensive step of drying the microorganisms.

SUMMARY OF THE INVENTION

The present invention provides a process for obtaining lipid frommicroorganisms comprising:

-   -   (a) lysing cells of the microorganisms to produce a lysed cell        mixture;    -   (b) treating the lysed cell mixture to produce a phase separated        mixture comprising a heavy layer and a light layer;    -   (c) separating the heavy layer from the light layer; and    -   (d) obtaining the lipid from the light layer.        The lysed cell mixture may contain an emulsion, in which case        the emulsion can be separated by centrifuging the lysed cell        mixture. The separated lysed cell mixture comprises a heavy        layer which contains aqueous solution and a light layer which        contains lipids, which may be emulsified. The aqueous solution        comprises solid cell materials which results from lysing cells.        The light layer can be further washed with an aqueous washing        solution until the lipid becomes substantially non-emulsified.

When the lipid extraction process of the present invention includesusing microorganisms from a fermentation process, the extraction processcan also include solubilizing at least part of proteinaceous compoundsin a fermentation broth, by adding a base selected from the groupconsisting of hydroxides, carbonates, bicarbonates and mixtures thereof.

The process of the present invention can also include heating themicroorganisms to temperature of at least about 50° C.

Preferably, the microorganisms are capable of growth at salinity levelof less than about 12 g/L of sodium chloride, more preferably less thanabout 5 g/L of sodium chloride and most preferably less than about 3 g/Lof sodium chloride.

Preferably, the microorganisms comprise at least about 30% by weight oflipid, more preferably at least about 35% by weight, and most preferablyat least about 40%. Alternatively at least about 30% of the lipid isdocosahexaenoic acid, preferably at least about 35%, and more preferablyat least about 40%.

In one particular aspect of the present invention the microorganisms arecapable of producing at least about 0.1 grams per liter per hour ofdocosahexaenoic acid, more preferably at least about 0.2 g/L/h, stillmore preferably at least about 0.3 g/L/h, and most preferably at leastabout 0.4 g/L/h.

In another aspect of the present invention, the microorganism isselected from the group consisting of algae, fungi, bacteria andprotist. Preferably, the microorganisms are of the orderThraustochytriales. More preferably the microorganisms are selected fromthe genus Thraustochytrium, Schizochytrium and mixtures thereof. Andmost preferably, the microorganisms are selected from the groupconsisting of microorganisms having the identifying characteristics ofATCC number 20888, ATCC number 20889, ATCC number 20890, ATCC number20891 and ATCC number 20892, mutant strains derived from any of theforegoing, and mixtures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of one embodiment of a solventless extractionprocess of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a process for extracting,recovering, isolating or obtaining lipids from microorganisms. Theprocess of the present invention is applicable to extracting a varietyof lipids from a variety of microorganisms, for example, extractinglipids containing omega-3 highly unsaturated fatty acids, such asdocosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and/ordocosapentaenoic acid (DPA), in particular lipids containing arelatively large amount of DHA, from microorganisms producing the sameand extracting lipids containing arachidonic acid from microorganismsproducing the same. Exemplary microorganisms which produce a relativelylarge amount of omega-3 highly unsaturated fatty acids are disclosed incommonly assigned U.S. Pat. Nos. 5,340,594 and 5,340,742, both issued toBarclay, and exemplary microorganisms which produce a relatively largeamount of arachidonic acid are disclosed in commonly assigned U.S. Pat.No. 5,583,019, issued to Barclay. All of the above disclosed patents areincorporated herein by reference in their entirety.

For the sake of brevity, however, this detailed description of theinvention is presented for purposes of convenience and illustration forthe case of extracting lipids comprising omega-3 highly unsaturatedfatty acid from microorganisms producing the same, in particularextracting lipids from microorganisms which produce a relatively highamount of DHA. It is to be understood, however, that the invention as awhole is not intended to be so limited, and that one skilled in the artwill recognize that the concept of the present invention will beapplicable to other microorganisms producing a variety of lipidcompositions in accordance with the techniques discussed herein. Thesemicroorganisms include microorganisms, such as fungi, protist, algae andbacteria, which produce a variety of lipids, such as phospholipids; freefatty acids; esters of fatty acids, including triglycerides of fattyacids; sterols; pigments (e.g., carotenoids and oxycarotenoids) andother lipids, and lipid associated compounds such as phytosterols,ergothionine, lipoic acid and antioxidants including beta-carotene andtocopherol. Exemplary lipids include, but are not limited to,arachidonic acid, stearidonic acid, cholesterol, desmesterol,astaxanthin, canthaxanthin, and n-6 and n-3 highly unsaturated fattyacids such as eicosapentaenoic acid, docosapentaenoic acid anddocosahexaenoic acid. For the sake of brevity, unless otherwise stated,the term “lipid” refers to lipid and/or lipid associated compounds.Other lipids and microorganisms which may be suitable for use in theinstant invention will be readily apparent to those skilled in the art.

Typical microbial lipid (in particular an oil containing an omega-3highly unsaturated fatty acid such as DHA) manufacturing processesinvolve growing microorganisms which produce DHA in a fermentor,isolating the microorganisms, and extracting the intracellular lipidswith organic solvent, e.g., hexane. The extracted lipid is generallyfurther refined to produce a high purity and/or quality lipid. Theisolation of microorganisms involves diluting the fermentation brothwith water and centrifuging the mixture to isolate microorganisms. Whenlipids are not extracted immediately or soon after isolating themicroorganisms, the isolated microorganisms are typically dried, forexample, on a drum dryer, and sealed in a package, e.g., invacuum-sealed bags, to prevent degradation of lipids. Unfortunately, thedrying process exposes the microorganisms to heat, which can damage,i.e., degrade the quality of, the lipid if done incorrectly. The packagemay develop leaks, which can further degrade the quality of the lipids.Furthermore, if the dried microorganisms are not treated with anantioxidant, the exposure of microorganisms to air can further degradelipids.

Recovering the crude oil directly from the fermentation broth avoidsthese problems. Avoiding the organic solvent extraction step reducesmanufacturing costs and also eliminates operator exposure to the driedmicroorganisms, which can cause an allergic response in someindividuals.

The present invention provides a method for obtaining lipids frommicroorganisms using a substantially organic solvent free extractionprocess, i.e., a “solventless” extraction process. The term “solventlessextraction process” refers to an extraction process which when anaqueous solvent is used, the aqueous solvent comprises less than about5% of an organic solvent, preferably less than about 4%, morepreferablyless than about 2%, and most preferably less than 1%. Theprocess of the present invention can include obtaining or isolatingmicroorganisms, preferably from a fermentation process. In contrast tothe current methods, the process of the present invention does notrequire a drying step prior to the extraction process. Thus, processesof the present invention are applicable to extracting lipids from amicrobial biomass containing at least about 10% by weight entrainedwater, preferably at least about 20%, more preferably at least about30%, and most preferably at least about 50%. When the microorganisms areobtained from a fermentation process, the process of the presentinvention can include adding a base to the fermentation broth todissolve any proteinaceous compound that may be present in the broth. A“base” refers to any compound whose pKa is greater than that of water.The base should be strong enough to hydrolyze at least a portion ofproteinaceous compounds that may be present in the broth. Bases whichare useful for solubilizing proteins are well known to one of ordinaryskill in the art of chemistry. Exemplary bases which are useful in theprocesses of the present invention include, but are not limited to,hydroxides, carbonates and bicarbonates of lithium, sodium, potassium,calcium, and magnesium carbonate.

The process of the present invention can also include rupturing orlysing the cells of microorganisms to release the lipids which arepresent within the cells. Cells can be lysed using any of the knownmethods including chemical; thermal; mechanical, including, but notlimited to, french press, mills, ultrasonication, and homogenization;and combinations thereof. In a thermal lysing of cells, the fermentationbroth containing microorganisms are heated until cells, i.e., cellwalls, of microorganisms degrade or breakdown. Typically, thefermentation broth is heated to a temperature of at least about 50° C.,preferably at least about 75° C., more preferably to at least about 100°C., and most preferably to at least about 130° C. Thermally lysing thecell walls of microorganisms is particularly useful for microorganismswhose cell walls are composed of proteins.

Heating the broth also denatures proteins and helps solubilize organicmaterials, including proteins. Heating of the fermentation broth stepcan be achieved by any of the known methods, including the use of anin-line heat exchanger, and preferably by sparging steam into thefermentor and maintaining the broth at a desired temperature for lessthan about 90 minutes, preferably less than about 60 minutes, and morepreferably less than about 30 minutes.

The solventless extraction process of the present invention can alsoinclude at least partially separating the broth from lipids. Typically,this is achieved by centrifuging, e.g., by passing the broth through astacked-disc centrifuge, and collecting lipids as an emulsion phase.Centrifuging the mixture results in a two phase mixture comprising aheavy layer and a light layer. Typically, the heavy layer is an aqueousphase, which contains the majority of cellular debris. The light layerwhich contains emulsified lipids is then diluted with water, againseparated into two phase mixture and the light layer is again isolated.This dilution with water, separation and isolation processes (i.e.,washing process) can be achieved continuously by feeding water andremoving the heavy layer throughout the process or it can be conductedin discreet steps. The washing process is generally repeated until anon-emulsified lipid layer is obtained. It is believed that theoil-water interface of the emulsion is stabilized by residual cellulardebris which is removed by the washing process. During the washingprocess, the successive amount of water added is reduced to increase thelipid content. While reducing the amount of feed water too quickly canresult in loss of lipids to the aqueous phase, reducing the amount offeed water too slowly results in an inefficient washing process. One canreadily determine an appropriate rate of feed water reduction byobserving or analyzing the separated aqueous layer. Generally, the lipidlayer, i.e., the light layer, is colored; therefore, in many cases onecan determine an appropriate rate of feed water reduction by simplyanalyzing or observing the color of the aqueous layer which is separatedfrom the lipid layer.

The isolated lipid can be further refined using a process similar tothat used to refine standard vegetable oils. Briefly, the lipid refiningprocess generally involves hydrating phospholipids by adding phosphoricacid to the lipid followed by adding sodium hydroxide to neutralize freefatty acids. These compounds are removed via centrifugation. This isthen followed by a water wash step to further remove any remainingamounts of hydrated phospholipids (“gums”) and neutralized fatty acids(“soapstock”) in the lipid. The resulting lipid is bleached usingTrysil™ and a standard bleaching clay. Citric acid is also added toremove divalent metal ions by chelation. The Trysil™ and bleaching clayare then removed via filtration to produce refined lipid. The bleachedlipid can be cold filtered to remove high melting point compounds thatmay be present in the lipid; however, this step is generally seldomrequired.

The resulting lipid can be further refined by removing any low molecularweight components that may be present. Typically, these components areremoved by sparging with steam at high temperatures, under high vacuum.This process also destroys any peroxide bonds which may be present andreduces or removes off odors and helps improve the stability of the oil.An antioxidant may then be added to the resulting deodorized lipid toimprove product stability.

Prior to the refining process, the isolated lipid can be winterized toremove high melting compounds, such as saturated fatty acids. Thewinterization process generally involves dissolving the isolated lipidin an organic solvent, e.g., hexane, cooling the resulting organicsolution, and filtering the solution to remove the high melting pointcomponents of the lipid or stearine phase. The winterization processgenerally produces a clear lipid, especially when the isolated lipid iscloudy or opaque.

While, the process of the present invention can include isolatingmicroorganisms from a fermentation process, one of the advantages of thepresent invention is that it allows fermentation of microorganisms andisolation of lipids to be carried out in a single vessel. For example,after the fermentation, one can add base to the fermentation vessel andheat the mixture to lyse cells. After separating the phase into a heavylayer and a light layer, the light layer can be transferred to anothervessel for further processing or the heavy layer can be removed from thefermentation vessel, for example, by draining through the bottom of thefermentation vessel, and the remaining light layer can be furtherprocessed within the same fermentation vessel.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES

Process reproducibility was characterized by producing three samples offully refined oil using crude oil from the new solventless extractionprocess. A hexane-extracted sample was also fully refined to serve as acontrol. The fermentation, extraction and oil isolation steps wereperformed at a large scale, while the oil refining studies wereperformed at a small scale.

The fully refined oil samples were analyzed to demonstrate processreproducibility.

Fermentation:

A single F-Tank batch (˜1,200 gallons) was used to generate the startingbroth for the three solventless extraction processes. The batch(#F99202) was allowed to run for 94 hours, while controlling the glucoselevels at 13 g/L, after which time the corn syrup feed was terminated.Residual glucose levels dropped to <5 g/L four hours later. Thisresulted in a final age of 98 hours. The final broth volume was 958gallons. The final yield was 146 g/L. Both in-process contaminationchecks and a thorough analysis of a final broth sample failed to showany signs of contamination.

Hexane-Extracted Control Sample:

A small aliquot of broth from batch #F99202 was drum-dried and extractedwith hexane to serve as a control sample. The biomass intermediate(DHAINT Lot #9F0067A) was recovered using a 66 ft² double-drum dryer.Analysis of this lipid is shown in Table 1.

TABLE 1 Analysis of DHAINT Lot #9F0067A. Parameter Value DHA Content(FAME basis) 35.7% Oil Content 62.7% Peroxide Value (meq/kg) 2.6 TotalPlate Count (cfu/g) <50 DHA Content* 20.3% FAME Content 56.9% *cellulardry weight basisSolventless Extraction Process:

Crude oil was obtained by treating three 400-gallon aliquots (approx.)of broth in batch #F99202. Each 400-gallon aliquot from the F-Tank batchwas processed separately, starting with the caustic/heat treatmentsteps. Each aliquot was treated with 20 grams of 45% KOH per liter andheated to 130° C. for about 30 minutes by passing stream through thefermentation broth. The crude oil was recovered from the treated brothusing a commercial-scale Westfalia HFA-100 stacked-disc centrifuge.Summary results for various process parameters are reported in Table 2,and the final crude oil analysis results are shown in Table 3.

TABLE 2 Process Data from the Solventless Extraction Process. SFE-1SFE-2 SFE-3 Broth Treatment Volume of Broth Processed 288 gal 288 gal258 gal Final Treated pH 7.5 8.0 8.7 Final Volume After Heat Treatment388 gal 398 gal 308 gal Volume Increase From Condensate 34.7% 38.2%19.4% 1^(st) Pass Emulsion Total Volume (gal) 180 133 149 Est. OilConcentration (w/w) 12.0% 24.5% 16.1% Apparent Density (g/mL) 0.9860.991 0.999 Oil Isolation Total Crude Oil Recovered (lb) 182 165 174DHAOIL Lot Number Assigned 9F0001A 9F0002A 9F0003A

TABLE 3 Analysis of Lots of DHA from the Solventless Extraction Process.Parameter 9F0001A 9F0002A 9F0003A DHA Content (% FAME) 39.0% 38.6% 39.2%Peroxide Value (meq/kg) 4.6 1.8 2.0 Acid Value (mg KOH/g) N/D N/D N/DMoisture Content N/D N/D N/DRefining:

A sample from each aliquot of crude oil was winterized, refined,bleached and deodorized at a small scale, as was a sample of the crudeoil from the hexane-extracted control. Miscellaneous process data fromthese small scale experiments is shown in Table 4, including recoveryefficiencies for the various processing steps. While it is difficult toread too much into recovery efficiencies for bench-scale processes, aslosses tend to be disproportionately large, the values listed in Table 4show that values for the solventless-extracted samples tend to bracketthe values measured for the hexane-extracted control, with the oneexception being the winterization step. While the recovery efficiencyduring the winterization step for the hexane control was lower thanthose observed for the other three samples, this difference isinsignificant from a statistical perspective. The high losses during thewinterization step caused the overall recovery efficiency for thehexane-control sample to be lower as well. The lower yield would not beexpected to have a significant impact on the overall quality of the oil.All in all, differences in the processing of the various oil sampleswere minimal.

TABLE 4 Miscellaneous Process Data from the Oil Refining Steps. HEX-1SFE-1 SFE-2 SFE-3 Processing Conditions Miscella Concentration 45.0%52.9% 52.8% 45.0% Steam Sparge Rate 3.4% 3.4% 2.5% 2.2% RecoveryEfficiencies Winterization 80.6% 92.3% 87.7% 85.5% Refining 89.4% 84.8%91.8% 95.0% Water Wash 90.6% 94.5% 95.8% 81.2% Bleaching 86.1% 89.2%87.3% 84.1% Deodorization 97.4% 96.1% 97.2% 97.5% Packaging 88.2% 89.7%89.3% 95.8% Overall 48.2% 56.9% 58.5% 51.8% Final Product Lot Number9F0009A 9F0010A 9F0011A 9F0012AFully refined oil samples from the three solventless extraction runs,and the hexane-extracted control, were analyzed and the results areshown in Table 5. Also shown are the corresponding releasespecifications for each parameter.

A sample of the starting crude oil from the solventless extraction runwas also analyzed for iron content. The iron content of this sample(DHAOIL Lot #9F0002P) was 0.08 ppm. The concentration of the other tracemetals was all below their respective detection limits.

TABLE 5 QC Results for RBD Oil from the Solventless Extraction Process.Hexane Solventless Extraction Run ID # HEX-1 SFE-1 SFE-2 SFE-3 DHALIP-NSLot # 9F0009A 9F0010A 9F0011A 9F0012A Peroxide Value 0.28 0.69 0.35 0.34(meq/kg) Acid Value 0.17 0.11 0.57 0.24 (mg KOH/g) Moisture & Volatiles0.00% 0.06%** 0.00% 0.00% Trace Metals (ppm) Lead <0.20 <0.20 <0.20<0.20 Arsenic <0.20 <0.20 <0.20 <0.20 Iron 0.22 0.21 0.56*** 0.02 Copper<0.05 <0.05 <0.05 <0.05 Mercury <0.20 <0.20 <0.20 <0.20 DHA (% FAME)36.9 37.3 37.0 37.7 DHA (mg/g oil) 342 345 343 351 Hexane (ppm) <3 <3 <3<3 *Value was reduced to 0.22 mg KOH/g after repeating the refining andbleaching steps **Sample analyzed by the San Diego Fermentation SciencesAnalytical Group. ***Value was reduced to <0.02 ppm after repeating therefining and bleaching steps

Shown in Table 6 is a more direct comparison of the average analysisresults for the three samples from the solventless extraction processversus those for the hexane control.

TABLE 6 Comparison of Average Values. Hexane Solventless ExtractionParameter Control Mean Std Dev CV % Diff Peroxide Value  0.28  0.46 0.2043.3% 64.3% (meq/kg) Acid Value  0.17  0.19* 0.06 33.3% 11.2% (mg KOH/g)Moisture & 0.00% 0.02% 0.03%  173% ND Volatiles Trace Metals (ppm) Lead<0.20 <0.20 N/A N/A 0.0% Arsenic <0.20 <0.20 N/A N/A 0.0% Iron  0.22 0.26 0.27  104% 18.2% Copper <0.05 <0.05 N/A N/A 0.0% Mercury <0.20<0.20 N/A N/A 0.0% DHA Content 36.9% 37.3%  0.4%  0.9% 1.1% (% FAME) DHAContent 342  346   4    1.2% 1.2% (mg/g) Hexane (ppm) <3   <3   N/A N/A0.0% *Calculated using the acid value for the re-worked sample.

The results from this experiment clearly demonstrate that thesolventless extraction process is both reproducible and lipids fromsolventless extraction are relatively indistinguishable from the lipidsobtained from hexane extraction process in terms of process performanceand product quality. The final product from the solventless extractionprocess is substantially equivalent to lipids from a currenthexane-based extraction process, as determined by similarities betweenthe fatty acid and sterol profiles of the product from these twoprocesses.

The present invention, in various embodiments, includes components,methods, processes, systems and/or apparatus substantially as depictedand described herein, including various embodiments, subcombinations,and subsets thereof. Those of skill in the art will understand how tomake and use the present invention after understanding the presentdisclosure. The present invention, in various embodiments, includesproviding devices and processes in the absence of items not depictedand/or described herein or in various embodiments hereof, including inthe absence of such items as may have been used in previous devices orprocesses, e.g., for improving performance, achieving ease and\orreducing cost of implementation.

The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1. A process for obtaining lipids from microorganisms comprising: (a)growing said microorganisms in a fermentation medium to produce afermentation broth; (b) contacting said fermentation broth with a baseto dissolve at least a part of any proteins present in said fermentationbroth; (c) releasing intracellular lipids of cells of the microorganismsinto the fermentation broth by lysing the cells, rupturing the cells, orpermeabilizing the cells; (d) separating substances of differentdensities in the fermentation broth to produce a phase separated mixturecomprising a heavy layer and a light layer, wherein said heavy layercomprises an aqueous solution and said light layer comprises emulsifiedlipids; (e) removing said heavy layer from said phase separated mixture;(f) adding an aqueous washing solution to said light layer; (g)separating substances of different densities from said mixture of step(f); (h) removing said heavy layer from said phase separated mixture;and (i) repeating said steps (f)-(h) until said lipid becomessubstantially non-emulsified.
 2. The process of claim 1, wherein saidbase is selected from the group consisting of hydroxides, carbonates,bicarbonates, and mixtures thereof.
 3. The process of claim 1, whereinstep (d) comprises centrifuging the fermentation broth.
 4. The processof claim 1, wherein said aqueous solution of step (d) comprises solidcell materials.
 5. The process of claim 1, wherein said microorganism iscapable of growth at salinity level of less than about 12 g/L of sodiumchloride.
 6. The process of claim 1, wherein said microorganismcomprises at least about 30% by weight of lipid.
 7. The process of claim1, wherein said microorganism is selected from the group consisting ofalgae, fungi, bacteria and protist.
 8. The process of claim 1, whereinsaid microorganisms comprise microorganisms of the orderThraustochytriales.
 9. The process of claim 8, wherein saidmicroorganisms are selected from the genus Thraustochytrium,Schizochytrium and mixtures thereof.
 10. The process of claim 9, whereinsaid microorganisms are selected from the group consisting ofmicroorganisms having the identifying characteristics of ATCC number20888, ATCC number 20889, ATCC number 20890, ATCC number 20891 and ATCCnumber 20892, mutant strains derived from any of the foregoing, andmixtures thereof.
 11. The process of claim 1, wherein saidmicroorganisms are capable of producing at least about 0.1 grams perliter per hour of docosahexaenoic acid.
 12. The process of claim 1,wherein at least about 30% of said lipid is docosahexaenoic acid. 13.The process of claim 1, wherein the step of producing is conducted inless than about 5% of an organic solvent.
 14. The process of claim 13,wherein the organic solvent is hexane.
 15. The process of claim 1,wherein the step of producing is conducted in less than about 4% of anorganic solvent.
 16. The process of claim 1, wherein the step ofproducing is conducted in less than about 2% of an organic solvent. 17.The process of claim 1, wherein the step of producing is conducted inless than about 1% of an organic solvent.
 18. The process of claim 1,wherein the step of producing is conducted in the absence of an organicsolvent.
 19. The process of claim 1, wherein the releasing intracellularlipids comprises increasing the temperature of said fermentation brothto at least about 50° C. to lyse cells of the microorganisms to producea lysed cell mixture.